This proposal represents the continuation of our studies to dissect, in molecular detail, the process by which helper CD4+ T lymphocytes induce the differentiation of B lymphocytes that drives the humoral immune response. The progress from our lab and others has demonstrated that the CD4+ T cell surface protein, CD40-ligand (CD40-L)(previously termed 5c8 Ag or T-BAM in this grant) and its receptor, CD4O, play important roles in this process, because CD4O-L:CD4O interactions are essential for Ig isotype switching in vivo and their roles in other aspects of B cell differentiation have been suggested by a variety of in vitro studies. Central to further understanding this pathway is the recent identification by several groups, including our own, of a critical signalling element in the CD4O pathway, termed CD40-receptor associated factor 1 (CRAF1, LAP1, CD40bp). CRAF1 is a novel protein that binds functionally to CD4O cytoplasmic tail and contains both zinc ring and zinc finger domains, suggesting that CRAF1 may translocate to the nucleus to deliver CD4O signals. Therefore this grant focuses on the elucidation of the roles of CRAF1 in several aspects of CD4O-mediated signalling of B cells, including; CD23 upregulation, ICAM1 (CD54) upregulation and homotypic aggregation, upregulation of the costimulatory molecule CD8O (B7/BB-1) and Ig isotype switching. Since CD40 signalling plays an essential role in limiting apoptosis, detailed analysis of the role of CRAF1 in the rescue of B cells from Fas-mediated apoptosis is proposed. In addition, we propose to analyze the role of CRAF1 in patients with Hyper-IgM Syndrome (HIM) (high IgM, low or absent IgG, IgA and IgB) with normal CD4O-L because our preliminary analysis of two such unrelated individuals revealed similar premature termination codons in CRAF1 alleles that predict truncated CRAF1 peptides which lack the domain that binds the cytoplasmic tail of CD40. Moreover, cells from these HIM patients and cDNA clones derived from PCR cloning of their alleles provide additional resources to study the mechanism of CRAF1 signalling. Furthermore, we propose to characterize alternative splicing events, particularly in the 5'-region of CRAF1, which may reveal alternative isoforms of CRAF1-encoded peptides. Together, the proposed studies should advance our understanding of T cell helper function and its role in immunity and the maintenance of human health.